The present invention relates generally to materials comprising chitin-binding fragments of human chitinase enzyme and analogs of the fragments. More particularly, the invention relates to novel purified and isolated polynucleotides encoding such fragment products, to the chitinase fragment products encoded by such polynucleotides, to materials and methods for the recombinant production of such chitinase fragment products and to therapeutic and diagnostic uses of such chitinase fragment products.
Chitin is a linear homopolymer of xcex2-(1,4)-linked N-acetylglucosamine residues. This polysaccharide is second only to cellulose as the most abundant organic substance. The exoskeleton of arthropods is composed of chitin. In addition, fungi and other parasites contain chitin in their outer cell wall, where it serves important structural and protective roles. Disruption of the fungal cell wall and membrane has been a useful therapeutic strategy against fungi and parasites. For example, Amphotericin B and fluconazole exert their anti-fungal activity by affecting membrane steroids. Despite the existence of anti-fungal therapeutics, fungal infections of humans have increasingly become responsible for life-threatening disorders. See, Georgopapadakou et al., Trends Microbiol., 3:98-104 (1995). The fungal species and parasites responsible for these diseases are mainly Candida, Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chemotherapy, and immunosuppressed organ transplant patients.
Chitin can be degraded by the enzyme chitinase. Chitinase enzymes are found in plants, microorganisms, and animals. Bacterial chitinase helps to provide a carbon source for bacterial growth. Insects produce chitinase to digest their cuticle at each molt. In plants, chitinase is thought to provide a protective role against parasitic fungi. Chitinases have been cloned from numerous bacterial [e.g., Serratia marcescens, Jones et al., EMBO J., 5:467-473 (1986)], plant [e.g., tobacco, Heitz et al., Mol. Gen. Genet., 245:246-254 (1994)], and insect [e.g., wasp, Krishnan et al., J. Biol. Chem., 269:20971-20976 (1994)] species and have been categorieed into two distinct families, designated family 18 and family 19, based on sequence similarities [Henrissat and Bairoch, Biochem, J. 293:781-788 (1993)]. Although the catalytic region of the enzymes in family 18 is largely conserved across numerous species, there is very limited sequence similarity across species for the chitin-binding domain. The only feature common to several family 18 chitin-binding domains is the presence of multiple cysteine residues.
Several proteins with low homology to bacterial, insect, and plant chitinases (less than 40% amino acid identity) have been identified in mammals, such as human cartilage gp-39 (C-gp39) [Hakala et al., J. Biol. Chem., 268:25803-25810 (1993)], human glycoprotein YKL-40 [Johansen et al., Eur. J. Cancer, 31A:1437-1442 (1995)], oviduct-specific, estrogen-induced protein from sheep [DeSouza et al., Endocrinology, 136:2485-2496 (1995)], cows and humans; and a secretory protein from activated mouse macrophages [Chang et al., Genbank M94584]. However, chitin-degrading activity has not been reported for these proteins. The function of these proteins is not known, but they have been postulated to be involved in tissue remodeling. Hakala et al., supra, report that C-gp39 is detectable in synovial and cartilage specimens from rheumatoid arthritis patients, but not from normal humans. Recklies et al., Arthritis Rheumatism, 36(9 SUPPL.):S190 (1993) report locaization of the C-gp39 protein to a distinct population of cells in the superficial layers of cartilage. Johansen et al., supra, report that measurements of YKL-40 serum levels are of value as a potential prognostic marker for the extent of metastatic disease and survival of patients with recurrent breast cancer.
Escott et al., Infect. Immun., 63:4770-4773 (1995) demonstrated chitinase enzymatic activity in human leukocytes and in human serum. Overdijk et al., Glycobiology, 4:797-803 (1994) described isolation of a chitinase (4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase) from human serum and rat liver. Renkema et al., J. Biol. Chem., 270:2198-2202 (February 1995) prepared a human chitotriosidase from the spleen of a Gaucher disease patient. Their preparation exhibited chitinase activity and the article reports a small amount of amino acid sequence of the protein component of the preparation (22 amino terminal residues and 21 residues of a tryptic fragment). The function of human chitinase is also unknown, but a relationship with the pathophysiology of Gaucher disease is proposed in the article. A later publication by the same group [Boot et al., J. Biol. Chem., 270(44):26252-26256 (November 1995)] describes the cloning of a human macrophage cDNA encoding a product that exhibits chitinase activity. The partial amino acid sequence reported by the group in their February 1995 article matches portions of the deduced amino acid sequence of the human macrophage cDNA product. See also International Patent Publication No. WO 96/40940, which reports two distinct human chitotriosidase cDNAs encoding a 50 kD and a 39kD product, both of which were fully enzymatically active. Renkema et al., Eur. J. Biochem., 244:279-285 (1997) reported that human chitinase is initially produced in macrophages as a 50 kD protein that is in part processed into a 39 kD form that accumulates in lysozymes, and also reported that alternative splicing generates a distinct human chitinase mRNA species encoding a 40 kD chitinase. Both the 39 kD and 40 kD isoforms appeared to be C-terminally truncated and displayed full chitinase enzymatic activity but bound chitin poorly.
In view of the increasing incidence of life-threatening fungal infection in immunocompromised individuals, there exists a need in the art to identify new materials and methods useful for diagnosing and treating fungal infections.
The present invention provides novel purified and isolated polynucleotides (i.e., DNA and RNA, both sense and antisense strands) encoding human chitinase fragments and analogs thereof having chitin-binding activity but lacking chitinase enzymatic activity; methods for the recombinant production of such fragment products; purified and isolated human chitinase polypeptide fragment products; pharmaceutical compositions comprising such fragment products; and diagnostic or therapeutic agents conjugated to such fragment products thereof. Such fragment products and diagnostic or therapeutic agents conjugated thereto are expected to be useful for detecting chitin, binding chitin, and treating fungal infections or for development of products useful for treating fungal infections.
The nucleotide sequence of two human cDNAs encoding presumed allelic variants of human chitinase, and including noncoding 5xe2x80x2 and 3xe2x80x2 sequences, are set forth in SEQ ID NO: 1 and SEQ ID NO: 3. The human chitinase coding region corresponds to nucleotides 2 to 1399 of SEQ ID NO: 1 or nucleotides 27 to 1424 of SEQ ID NO: 3, and the putative coding sequence of the mature, secreted human chitinase protein without its signal sequence corresponds to nucleotides 65 to 1399 of SEQ ID NO: 1, or nucleotides 90 to 1424 of SEQ ID NO: 3. The amino acid sequences of the polypeptides encoded by the DNA of SEQ ID NOS: 1 and 3 are set forth in SEQ ID NO:2 and SEQ ID NO: 4, respectively. Twenty-one amino-terminal amino acids (positions xe2x88x9221 to xe2x88x921 of SEQ ID NOS: 2 and 4) comprise a signal peptide that is cleaved to yield the mature human chitinase protein (positions 1 to 445 of SEQ ID NOS: 2 and 4). It has been determined that the seventy-two C-terminal residues of human chitinase are not critical to chitinase enzymatic activity. Example 5 below illustrates production of an N-terminal fragment that lacks the seventy-two C-terminal residues of human chitinase; the introduction of a stop codon after the codon for amino acid 373 resulted in a recombinant chitinase fragment of about 39 kDa that retained similar specific chitinase enzymatic activity when compared with full length recombinant human chitinase. The cloning of human chitinase cDNA and expression thereof, and the biological activities of recombinant human chitinase are described in detail in U.S. application Ser. No. 08/877,599 filed Jun. 16, 1997, which is a continuation-in-part of U.S. application Ser. No. 08/663,618 filed Jun. 14, 1996, both of which are incorporated herein by reference in their entirety.
The present invention is based on the unexpected discovery that substantially all of the chitin-binding activity of human chitinase is contained within the 99 C-terminal amino acid residues of the 445 amino acid enzyme. Specifically provided by the present invention are chitin-binding, chitinase-inactive polypeptide products. Preferred chitinase fragment products comprise a chitin-binding fragment within the 54 C-terminal amino acids of human chitinase, including a fragment consisting of about the 99 C-terminal amino acids of human chitinase (about residues 347 through 445 of SEQ ID NO: 2), a fragment consisting of about the 72 C-terminal amino acids of human chitinase (about residues 374 through 445 of SEQ ID NO: 2), a fragment consisting of about the 54 C-terminal amino acids of human chitinase (about residues 392 through 445 of SEQ ID NO: 2), and a fragment consisting of about the 49 C-terminal amino acids of human chitinase (about residues 397 through 445 of SEQ ID NO: 2). Also provided by the invention are purified, isolated polynucleotides including DNA encoding such polypeptide fragments; vectors comprising such DNAs, particularly expression vectors wherein the DNA is operatively linked to an expression control DNA sequence; host cells stably transformed or transfected with such DNAs in a manner allowing the expression in said host cell of human chitinase fragment products; a method for producing human chitinase polypeptide fragment products comprising culturing such host cells in a nutrient medium and isolating such polypeptides from said host cell or said nutrient medium; purified, isolated polypeptides produced by this method; fusion proteins comprising such polypeptides fused to a heterologous peptide or polypeptide, including an enzyme such as secreted alkaline phosphatase (SEAP); compositions comprising such human polypeptide fragment products; compositions comprising a human chitinase polypeptide fragment product conjugated to an anti-fungal agent and methods of treating fungal infection by administering such compositions, optionally with co-administration of additional non-chitinase anti-fungal agents; compositions comprising a chitinase polypeptide fragment product conjugated to a detectable label (including radioisotopes, fluorophores, dyes, electron-dense compounds and enzymes), methods for using such compositions to determine the presence or amount of chitin in a sample, comprising the steps of: (a) contacting the sample with a human chitinase polypeptide fragment product conjugated to a detectable label, and (b) determining the amount of labelled fragment product bound to chitin, and corresponding kits for diagnosing the presence of chitin in a sample; monoclonal antibodies that specifically bind a chitin-binding, chitinase-inactive fragment of human chitinase, including antibodies that specifically bind to an epitope within the 54 C-terminal amino acids of human chitinase as set forth in SEQ ID NO:2; and preferred monoclonal antibodies 243Q and 243M, and antibodies that compete with or bind to the same epitope as 243Q and 243M.
Chitinase polypeptide fragment products of the invention include fragments of human chitinase or allelic variants thereof that substantially retain chitin-binding activity without retaining substantial chitinase enzymatic activity, analogs of such fragments, and fusion proteins comprising such fragments or analogs. Chitinase polypeptide fragment products are useful in therapeutic and diagnostic applications as described below.
Among the xe2x80x9cchitin-binding domainxe2x80x9d fragments contemplated by the invention are those represented by amino acid residues X through Y of SEQ ID NO: 2, wherein X is a consecutive integer from 347 through 397 and Y is 445, and portions thereof that retain chitin-binding activity. One preferred fragment consists of the ninety-nine C-terminal amino acids of human chitinase (residues 347 through 445 of SEQ ID NO: 2); this fragment has been shown in Example 7 below to retain 80% of the chitin-binding activity of the mature chitinase. Yet other preferred fragments are the fifty-four C-terminal amino acids of human chitinase (residues 392 through 445 of SEQ ID NO: 2), and the 49 C-terminal amino acids of human chitinase (residues 397 through 445 of SEQ ID NO:2), which have also been shown in Example 7 to retain chitin-binding activity. As illustrated in Example 7, a fusion protein containing the 99 C-terminal amino acids of human chitinase was shown to contain the chitin-binding domain of the protein. The boundaries of the chitin-binding domain were further defined by N-terminal and C-terminal truncation of this 99 amino acid region and determination of the chitin binding activity of fusion proteins comprising these truncates. These truncates included those with an N-terminus commencing at amino acid residue 347, 374, 392, 395, 397, 400 or 409 and with a C-terminus at amino acid residue 431, 443 or 445.
Analogs may comprise chitinase fragment analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more nonspecified amino acids are added: (1) without loss of one or more of the biological activities (including chitin-binding activity) or immunological characteristics specific to chitinase; or (2) with specific disablement of a particular biological activity of chitinase. The invention contemplates that conservative amino acid substitutions as known in the art may be made without affecting the biological activity of the fragment.
Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences encoding chitin-binding fragments of human chitinase without chitinase enzymatic activity, analogs thereof, and fusion proteins comprising such fragments or analogs. Among the nucleotide sequences contemplated by the invention are those encoding the amino acid sequences of positions X through Y of SEQ ID NO: 2, wherein X is a consecutive integer from 347 through 392 and Y is 445. Nucleotides 1238 through 1399 of SEQ ID NO: 1 (encoding residues 392 through 445 of SEQ ID NO: 2) are a particularly preferred DNA sequence of the invention. This DNA sequence and other DNA sequences which hybridize to the noncoding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, and which encode chitin-binding fragments of a chitinase, are also contemplated by the invention. Exemplary stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in 50% formamide and washing at 60xc2x0 C. in 0.1xc3x97SSC, 0.1% SDS. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide base content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Among the uses for the polynucleotides of the present invention are use as a hybridization probe, to identify and isolate non-human genomic DNA and cDNA encoding chitin-binding regions of proteins homologous to human chitinase; and to identify those cells which express chitin-binding portions of such proteins and the biological conditions under which such proteins are expressed.
In another aspect, the invention includes biological replicas (i.e., copies of isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention. Autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating polynucleotides encoding chitin-binding fragments of human chitinase, including any of the DNAs described above, are provided. Preferred vectors include expression vectors in which the incorporated chitinase fragment-encoding cDNA is operatively linked to an endogenous or heterologous expression control sequence and a transcription terminator. Such expression vectors may further include polypeptide-encoding DNA sequences operably linked to the chitinase fragment-encoding DNA sequences, which vectors may be expressed to yield a fusion protein comprising the polypeptide of interest.
According to another aspect of the invention, procaryotic or eucaryotic host cells are stably transformed or transfected with polynucleotide sequences of the invention in a manner allowing the desired chitinase product to be expressed therein. Host cells expressing chitinase fragment products can serve a variety of useful purposes. Such cells constitute a valuable source of immunogen for the development of antibody substances specifically immunoreactive with chitinase. Host cells of the invention are useful in methods for the large scale production of chitinase fragment products wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated, e.g., by immunoaffinity purification, from the cells or from the medium in which the cells are grown.
Knowledge of DNA sequences encoding the chitin-binding portion of human chitinase allows for modification of cells to permit or increase expression of the chitin-binding portions. Cells can be modified, (e.g., by homologous recombination) to provide increased expression of the chitin-binding portion of human chitinase by inserting all or part of a heterologous promoter in the appropriate position within the gene. The heterologous promoter is inserted in such a manner that it is operably linked to the DNA sequence encoding the chitin-binding portion of human chitinase. See, for example, PCT International Publication Nos. WO 94/12650, WO 92/20808 and WO 91/09955. Amplifiable marker DNA and/or intron DNA may be inserted along with the heterologous promoter DNA.
Chitinase fragment products may be obtained as isolates from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving procaryotic or eucaryotic host cells of the invention. The use of mammalian host cells is also expected to provide for post-translational modifications (e.g., myristolation, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.
The invention further comprehends use of chitinase fragment products in screening for proteins or other molecules (e.g., small molecules) that specifically bind to the chitin-binding domain of human chitinase or that modulate binding of human chitinase to chitin or to human extracellular matrix proteins such as hyaluronic acid. Proteins or other molecules (e.g., small molecules) which specifically bind to chitinase can be identified using fragments of chitinase isolated from plasma, recombinant chitinase fragment products, or cells expressing such products. Proteins or other molecules that bind to the chitin-binding domain of chitinase may be used to modulate its activity. Binding proteins that specifically bind to the chitin-binding domain of chitinase are contemplated by the invention and include antibody substances (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, human antibodies, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention). By xe2x80x9cspecifically bind to the chitin-binding domain of chitinasexe2x80x9d it is meant that the binding protein recognizes exclusively the chitin-binding domain of chitinase and not the catalytically active portion of chitinase. Binding proteins are useful, in turn, in compositions for immunization as well as for purifying chitinase, and are useful for detection or quantification of chitinase in fluid and tissue samples by known immunological procedures. Anti-idiotypic antibodies specific for chitinase-specific antibody substances are also contemplated.
Antibodies that specifically bind to chitin-binding domain are useful in methods for detecting or quantifying the presence of chitin-binding domain, e.g., in a sandwich ELISA assay, and for detecting or quantifying the presence of yeast or fungi, e.g., by adding a chitin-binding domain which binds to the yeast or fungi, followed by adding a labeled antibody specific for the chitin-binding domain. Detection of chitin-binding domain in human blood (plasma or serum) samples may also be correlated to a diagnostic standard indicative of a disease state involving chitinase, such as Gaucher""s disease. Presently preferred antibodies are monoclonal antibodies 243Q and 243M, produced by hybridomas 243Q (Accession No. HB-12688) and 243M (Accession No. HB-12657), respectively, and monoclonal antibodies that compete with or bind to the same epitope recognized by 243Q or 243M.
The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for chitinase makes possible the isolation by DNA/DNA hybridization or polymerase chain reaction (PCR) of genomic DNA sequences encoding other mammalian chitinases and the like. DNA/DNA hybridization or PCR procedures carried out with DNA sequences of the invention under conditions of stringency standard in the art are likewise expected to allow the isolation of DNAs encoding human allelic variants of chitinase, other structurally related human proteins sharing the chitin-binding property of chitinase, and the chitin-binding regions of non-human species proteins homologous to chitinase. The DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or xe2x80x9cknockoutxe2x80x9d strategies [see, e.g., Kapecchi, Science, 244: 1288-1292 (1989)], of animals that fail to express a functional chitinase enzyme, overexpress chitinase enzyme, or express a variant chitinase enzyme. Such animals are useful as models for studying the in vivo activity of chitinase or modulators of chitinase. Polynucleotides of the invention when suitably labelled are useful in hybridization assays to detect the capacity of cells to synthesize chitinase. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in the chitinase locus that underlies a disease state or states. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of chitinase by those cells which ordinarily express the same.
The invention contemplates that chitin-binding fragment products may be fused to a heterologous polypeptide. For example, such products may be fused to a portion of an immunoglobulin, such as the constant region, for therapeutic purposes. As another example, such products may be fused to a polypeptide useful as a detectable label or marker, such as a polypeptide with enzymatic activity or a polypeptide carrying a specifically detectable epitope, such as a myc epitope or FLAG epitope tag (Eastman Kodak).
Chitin-binding fragments may also be fused to another protein of interest to facilitate purification of the protein of interest via affinity binding to a chitin matrix. The fusion protein may then be obtained by elution from the column, or the protein of interest may be cleaved from the chitin-binding domain followed by elution of the cleaved protein. See Chong et al., Gene, 192:271-281 (1997).
The human chitinase fragment products of the invention are also useful as a chitin-specific reagent for specifically identifying the presence of chitin in a sample. According to this aspect of the invention, a chitinase fragment product having chitin-binding activity is conjugated with a detectable label, such as a radioisotope, fluorophore, dye, electron-dense compound, or enzyme, contacted with the sample to be tested, and analyzed qualitatively or quantitatively for the presence of chitin. xe2x80x9cConjugatedxe2x80x9d as used herein means linked by covalent bonds. Such techniques are well known and illustrated in, e.g., U.S. Pat. No. 5,587,292, incorporated herein by reference. The amount of chitin thus measured can be indicative of the fungal load in an infected patient. Two preferred fragments for use according to this method are the 54 amino acid chitin-binding domain consisting of amino acid residues 392 through 445 of the human chitinase amino acid sequence set out in SEQ ID NO: 2 and the 49 amino acid chitin-binding domain consisting of amino acid residues 397 through 445 of SEQ ID NO: 2.
The invention also provides conjugates comprising chitin-binding chitinase fragments and an imaging agent, such as gamma- and positron-emitting radioisotopes for radionuclear imaging (e.g., 157Gd, 55Mn, 162Dy, 52Cr, 56Fe, 111In, 97Ru, 67Ga, 68Ga, 72As, 89Zr, 201Tl, 99Tn, 90Y); paramagnetic metal chelates, nitroxyl spin labelled compounds or other agents (e.g., Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III) and V(IV), GdDTPA/dimeglumine [Magnevist(trademark)]) for MRI imaging; contrast enhancement agents for X-ray based imaging, including CT scans (e.g., bromine- or iodine-containing compounds); and other agents known in the art. Such conjugates are expected to bind yeast cell wall chitin and thus to be useful in methods for detecting or localizing yeast in vivo in mammals.
Administration of chitinase fragment products and therapeutic agents comprising such products to mammalian subjects, especially.humans, for the purpose of ameliorating disease states caused by chitin-containing parasites such as fungi is contemplated by the invention. Fungal infections (mycoses) such as candidiasis, aspergillosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis, histoplasmosis, cryptococcosis, chromoblastomycosis, sporotrichosis, mucormycosis, and the dermatophytoses can manifest as acute or chronic disease. Pathogenic fungi cause serious, often fatal disease in immunocompromised hosts. Cancer patients undergoing chemotherapy, immunosuppressed individuals, and HIV-infected individuals are susceptible to mycoses caused by Candida, Aspergillus, Pneumocystis carinii, and other fungi. Amphotericin B and fluconazole are useful therapeutics for fungal infections, but toxicity associated with these drugs causes serious adverse side effects that limit their usefulness. The mortality of systemic candidiasis is greater than 50% despite Amphotericin B treatment. Animal models for fungal infection are illustrated below in Examples 9 through 15 and have been described in the art.
Specifically contemplated by the invention are compositions comprising chitinase fragment products for use in methods for treating a mammal susceptible to or suffering from fungal infections. It is contemplated that the chitinase fragment products may be conjugated to other conventional anti-fungal agents, including amphotericin B and the structurally related compounds nystatin and pimaricin; 5-fluorocytosine; azole derivatives such as fluconazole, ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, terconazole, itraconazole and tioconazole; allylamines-thiocarbamates, such as tolnaftate, naftifme and terbinafme; griseofulvin; ciclopirox olamine; haloprogin; undecylenic acid; and benzoic acid. [See, e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, NY (1996).] According to this aspect of the invention, the chitin-binding fragment products serve as a vector to target known fungicidal or fungistatic compounds to pathogenic chitin-bearing fungi, and thus may improve the effectiveness of these conventional anti-fungal agents, perhaps by rendering the fungi more susceptible to their action. A reduction in the amount of conventional anti-fungal agent needed to exert the desired therapeutic effect may allow the drugs to be used at less toxic levels. The ability to selectively target fungi or yeast using a chitin-binding domain fragment also allows administration of such fragments conjugated to cytotoxic agents that are not themselves selectively anti-fungal. This aspect of the invention contemplates conjugation of chitin-binding chitinase fragments to any cytotoxic agent known in the art, including radioisotopes (such as 90Y, 188Re, 186Re, 199Au, 64Cu, 67Cu, 131I), toxins and chemotherapeutic agents, that would be effective against yeast. Suitable cytotoxic agents can be easily identified using methods known in the art. Using human chitinase chitin-binding domain for this purpose is more advantageous than using chitin-binding domains of chitinases of other species because human polypeptides are expected to be non-immunogenic in humans.
Chitin-binding domain fragments themselves may have anti-fungal effects through disruptive cross-linking of yeast cell wall, and may be co-administered alone or in combination with other anti-fungal agents. Multimeric chitin-binding domain fragments, which may be especially useful for this purpose, are contemplated by the invention, including multimeric fragments that have been covalently cross-linked by chemical means and recombinantly produced polypeptides comprising multiple chitin-binding domains linked in tandem. The administration of chitin-binding domain fragments, either monomeric or multimeric, may reduce the amount of co-administered anti-fungal agent necessary to exert a desired therapeutic effect.
Thus, the invention contemplates the use of chitinase fragment products in the preparation of a medicament for the prophylactic or therapeutic treatment of fungal infections.
Therapeutic/pharmaceutical compositions contemplated by the invention include chitinase fragment products, which may be conjugated to another therapeutic agent, and a physiologically acceptable diluent or carrier and may also include other anti-fungal agents. Dosage amounts indicated would be sufficient to supplement endogenous chitinase activity. For general dosage considerations see Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa. (1995). Dosages will vary between about 1 xcexcg/kg to 100 mg/kg body weight, and preferably between about 0.1 to about 20 mg chitinase/kg body weight. Therapeutic compositions of the invention may be administered by various routes depending on the infection to be treated, including via subcutaneous, intramuscular, intravenous, intrapulmonary, transdermal, intrathecal, topical, oral, or suppository administration.
The invention also contemplates that the overexpression of chitinase in Gaucher disease or at sites of inflammation (such as in rheumatoid arthritis) may have deleterious effects on the extracellular matrix and, in such disease settings, inhibitors of chitinase activity, including chitinase fragment products themselves or inhibitors of chitin-binding identified by the screening methods described above, may provide therapeutic benefit, e.g. by reducing remodeling or destruction of the extracellular matrix.
The human chitinase cDNA has been isolated from a macrophage cDNA library. Macrophages are known to be closely associated with rheumatoid arthritis lesions [Feldman et al., Cell, 85:307-310 (1996)], and macrophage products such as TNP-xcex1 are implicated in disease progression. A protein with homology to human chitinase, C-gp39, has been detected in the synovium and cartilage of rheumatoid arthritis patients. While the natural substrate for human chitinase is probably chitin from pathogenic organisms, the enzyme may also exhibit activity on endogenous macromolecules which form the natural extracellular matrix. For example, it has been suggested that hyaluronic acid, a major component of the extracellular matrix, contains a core of chitin oligomers. [Semino et al., Proc. Nat""l Acad. Sci., 93:4548-4553 (1996); Varki, Proc. Nat""l. Acad. Sci., 93:4523-4525 (1996).] Chitinase may therefore be involved in degradation of extracellular matrix in diseases such as rheumatoid arthritis. The role of chitinase may be determined by measuring chitinase levels and/or the effects of chitinase administration or chitinase inhibition in synovial fluid isolated from arthritic joints. Endogenous chitinase levels can be measured by enzymatic assay or with an antibody. Viscosity of synovial fluid can be measured before and after chitinase treatment; a decrease of viscosity associated with chitinase would be consistent with an endogenous chitinase substrate. Modulation of chitinase activity could thereby modulate the progression of joint destruction in rheumatoid arthritis.
Also contemplated by the invention are methods for screening for inhibitors of chitinase activity, which may be useful in the manner described in the preceding paragraph. A method for screening samples to identify agents that inhibit chitinase is reported in, e.g., WO 95/34678 published Dec. 21, 1995.